Exosomal compositions and methods for the treatment of disease

ABSTRACT

An exosomal composition is provided that comprises a therapeutic agent encapsulated by an exosome. The therapeutic agent can be a phytochemical agent, a chemotherapeutic agent, or a Stat3 inhibitor. Pharmaceutical compositions comprising the exosomal compositions are also provided. Methods for treating an inflammatory disease or a cancer are further provided and include administering an effective amount of an exosomal composition to a subject in need thereof to thereby treat the inflammatory disorder or the cancer.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/359,618, filed Mar. 20, 2019 (pending), is a continuation of U.S.application Ser. No. 15/917,151, filed Mar. 9, 2018 (abandoned), whichis a continuation of U.S. application Ser. No. 14/107,691, filed Dec.16, 2013 (abandoned), which is a continuation of U.S. application Ser.No. 13/576,907, filed Aug. 2, 2012 (abandoned), and which is a 371national stage application of International Application No.PCT/US2011/023747, filed Feb. 4, 2011, and which claims priority fromU.S. Provisional Application Ser. No. 61/424,875, filed Dec. 20, 2010,and U.S. Provisional Application Ser. No. 61/301,939, filed Feb. 5,2010, the entire disclosures of each of which are incorporated herein bythis reference.

GOVERNMENT INTEREST

This invention was made with government support under grant numbersR01CA137037 and R01AT004294 awarded by National Institutes of Health.The government has certain rights in the invention.

TECHNICAL FIELD

The presently-disclosed subject matter relates to exosomal compositionsand methods of using the same for the treatment of disease. Inparticular, the presently-disclosed subject matter relates to exosomalcompositions that comprise therapeutic agents encapsulated by an exosomeand are useful in the treatment of inflammatory disorders and cancer.

BACKGROUND

Fifty years ago, the nanotechnology concept was initially proposed and,in 2000, the National Nanotechnology Institute (NNI) definednanotechnology as “[t]he understanding and control of matter atdimensions of roughly 1 to 100 nm, where unique phenomena enable novelapplications.” The application of nanoscale or nanostructured materialin medicine has since been extended to objects with sizes of up to 1000nm and numerous nanoparticle materials, such as polymers, liposomes,metals, and carbon nanotubes, are now being examined as potentialtherapeutic agent delivery vectors. For instance, researchers havedeveloped or are currently developing methods by which small moleculedrugs, peptides, proteins, DNA and even siRNA molecules are packed intonanoparticles and then used to treat multiple fungal infections,inflammatory diseases, bone defects, and cancers.

Despite ongoing research into the use of nanoparticles as therapeuticagent-delivery vectors, however, in the fight against inflammatorydisorders and cancers, localized drug delivery and specific targetingare two major problems researchers and clinicians must still confront.Indeed, one of the most challenging issues in anti-cancer andanti-inflammatory therapy continues to be achieving delivery oftherapeutic agents to specific inflammatory cells and tumor cells invivo and then, upon delivery, having the particular therapeutic agentretain its activity. For example, in spite of the development oftherapeutic agents that preferentially target inflammatory and cancercells without harming normal tissues, the delivery of these agents tothe brain continues to be a major challenge because of difficulty inpenetrating the blood-brain barrier (51-56). Indeed, the furtherdevelopment of many therapeutic agents has been abandoned becausesufficient therapeutic agent levels in the brain could not be achievedvia the systemic circulation. Intranasal delivery has previouslyprovided a noninvasive method for delivering therapeutic agents to thebrain in some instances, but the quantities of therapeutic agents thatare able to be administered via that route and that are transporteddirectly from nose-to-brain continue to be very low (61-65).

Recently, efforts have been made to confront these problems via thefurther development of nanoparticle-therapeutic agent delivery systems.To date, however, the development of an efficient and specificnanoparticle-therapeutic agent delivery system has yet to be producedthat is able to locally deliver the agent to the target cells andtissues while retaining a sufficient biological activity.

SUMMARY

The presently-disclosed subject matter meets some or all of theabove-identified needs, as will become evident to those of ordinaryskill in the art after a study of information provided in this document.

This Summary describes several embodiments of the presently-disclosedsubject matter, and in many cases lists variations and permutations ofthese embodiments. This Summary is merely exemplary of the numerous andvaried embodiments. Mention of one or more representative features of agiven embodiment is likewise exemplary. Such an embodiment can typicallyexist with or without the feature(s) mentioned; likewise, those featurescan be applied to other embodiments of the presently-disclosed subjectmatter, whether listed in this Summary or not. To avoid excessiverepetition, this Summary does not list or suggest all possiblecombinations of such features.

The presently-disclosed subject matter includes exosomal compositionsand methods of using the exosomal compositions for the treatment ofdisease. More specifically, the presently-disclosed subject matterrelates to exosomal compositions where one or more therapeutic agentsare encapsulated within an exosome and are used to treat inflammatorydisorders or cancers.

In some embodiments of the presently-disclosed subject matter, anexosomal composition is provided where an exosome encapsulates atherapeutic agent that is selected from a phytochemical agent, achemotherapeutic agent, and a Stat3 inhibitor. In some embodiments, thetherapeutic agent is a phytochemical agent selected from the groupconsisting of curcumin, resveratrol, baicalein, equol, fisetin, andquercetin. In other embodiments, the therapeutic agent is a Stat3inhibitor, such as JSI-124. In further embodiments, the therapeuticagent is a chemotherapeutic agent that, in certain embodiments, isselected from the group consisting of retinoic acid, 5-fluorouracil,vincristine, actinomycin D, adriamycin, cisplatin, docetaxel,doxorubicin, and taxol.

In some embodiments, the exosomal compositions that are produced inaccordance with the presently-disclosed subject matter make use ofexosomes that are first isolated from a cell before the exosomes arethen used to encapsulate a therapeutic agent of interest. In someembodiments, the exosomes are first isolated from a cancer cell, which,in some embodiments, is selected from a lymphoma cell, an adenocarcinomacell, or a breast cancer cell. In some embodiments, the exosomes arefruit-derived exosomes, such as grape exosomes.

Further provided, in some embodiments, are pharmaceutical compositionscomprising an exosomal composition of the presently-disclosed subjectmatter. In some embodiments, a pharmaceutical composition is providedthat comprises an exosomal composition of the presently-disclosedsubject matter and a pharmaceutically-acceptable vehicle, carrier, orexcipient.

Still further provided, in some embodiments of the presently-disclosedsubject matter, are methods for treating an inflammatory disorder. Insome embodiments, a method for treating an inflammatory disorder isprovided that comprises administering to a subject in need thereof aneffective amount of an exosomal composition of the presently-disclosedsubject matter. In some embodiments of the methods for treating aninflammatory disorder, the therapeutic agent is selected from aphytochemical agent and a Stat3 inhibitor. In some embodiments, theexosomal composition, which includes the phytochemical agent or theStat3 inhibitor encapsulated in an exosome, is administeredintranasally.

In some embodiments of the presently-disclosed subject methods fortreating an inflammatory disorder, the inflammatory disorder is abrain-related inflammatory disorder. In some embodiments, theinflammatory disorder is an autoimmune disease such as, in someembodiments, lupus, rheumatoid arthritis, or autoimmuneencephalomyelitis. In some embodiments, administering the exosomalcomposition as part of a method for treating an inflammatory disorderreduces an amount of an inflammatory cytokine in a subject. In someembodiments, the inflammatory cytokine is selected from the groupconsisting of interleukin-1β, tumor necrosis factor-α, andinterleukin-6.

In yet further embodiments of the presently-disclosed subject matter,methods for treating a cancer are provided. In some embodiments, amethod for treating a cancer is provided that comprises administering toa subject n need thereof an effective amount of an exosomal compositionof the presently-disclosed subject matter. In some embodiments of themethods for treating a cancer, the therapeutic agent encapsulated by anexosome is selected from a phytochemical agent, a chemotherapeuticagent, and a Stat3 inhibitor.

In some embodiments, the methods for treating a cancer disclosed hereinare used to treat a skin cancer, a head and neck cancer, a colon cancer,a breast cancer, a brain cancer, and a lung cancer. In some embodiments,the cancer is a brain cancer that, in some embodiments, comprises aglioma. In some embodiments, the exosomal compositions of thepresently-disclosed subject matter are used to treat the cancer byadministering the exosomal compositions intranasally, orally, orintratumorally.

Further advantages of the presently-disclosed subject matter will becomeevident to those of ordinary skill in the art after a study of thedescription, Figures, and non-limiting Examples in this document.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A includes photographs showing the results of sucrose gradientcentrifugation procedures used to purify exosomes encapsulating curcumin(i.e., exosomal curcumin), where the left image shows the originalexosomes without the encapsulated therapeutic agent as a weak band andthe right image shows the exosomal curcumin as a darker band appearingbetween higher concentration sucrose gradients;

FIG. 1B includes electron microscopy images showing the morphology andsize of exosomes isolated from an EL4 mouse lymphoma cell line (“EL4exo”; upper image) as compared to EL4 exosomes that encapsulate curcumin(“Exosomal cur”; lower image);

FIG. 1C includes images of a western blot analysis of EL4 exosomes andexosomal curcumin protein expression, where the upper image shows thepresence of the exosomal protein TSG101 and the lower image shows thepresence of the exosomal protein CD81;

FIG. 2A is a spectrograph used to measure the concentration of freecurcumin in phosphate-buffered saline (PBS) in control samples;

FIG. 2B is a spectrograph used to measure the concentration of curcuminencapsulated in EL4 exosomes;

FIG. 2C is a graph showing the degradation of curcumin and exosomalcurcumin after the incubation of samples at 37° C., where concentrationsof curcumin and exosomal curcumin were measured at 30, 60, 90, 120, and150 min;

FIG. 3A is a graph showing the concentration of curcumin and exosomalcurcumin at various time points in the plasma of mice that wereintraperitoneally injected with 100 mg/kg body weight of curcumin orexosomal curcumin;

FIG. 3B includes chromatograms from high-performance liquidchromatography experiments that were performed with blood samples ofmice, where the samples were taken 30 minutes after intraperitoneallyinjecting the mice with 100 mg/kg body weight of curcumin or exosomalcurcumin;

FIG. 4 is a graph showing the amounts of interleukin-6 (IL-6) and tumornecrosis factor-alpha (TNF-α) in cell culture supernatants, where thecells where treated with PBS, EL4 exosomes, exosomal curcumin, or freecurcumin;

FIG. 5A is a graph showing the percent of surviving mice subsequent tothe administration of lipopolysaccharide (LPS) and PBS, EL4 exosomes,exosomal curcumin, or free curcumin;

FIG. 5B is a graph showing the serum levels of IL-6 and TNF-α in micesubsequent to the administration of lipopolysaccharide (LPS) and PBS,EL4 exosomes, exosomal curcumin, or free curcumin;

FIG. 6A includes an image and a graph showing the fluorescence activatedcell sorter (FACS) analysis of CD11b⁺Gr1⁺ cells in leukocytes that wereisolated from the lungs of mice who were administered PBS, EL4 exosomes,exosomal curcumin, or free curcumin;

FIG. 6B is a graph showing the cellular curcumin concentration in Gr1⁺cells isolated from bone marrow cells and then treated with the sameconcentration of either free curcumin or exosomal curcumin;

FIG. 6C is a graph depicting the extent of cell death in Gr1⁺ cellstreated with 5 μM or 10 μM of free curcumin or exosomal curcumin, wherethe cells were treated for 8 hours and then stained with Annexin V-FITC;

FIG. 7A includes images showing the distribution offluorescently-labeled exosomes in the brains of mice, where the imageswere taken 30 min after the exosomes were intranasally administered tothe mice, and where the exosomes were isolated from various types ofcells;

FIG. 7B includes images showing the distribution offluorescently-labeled exosomes in the brains of mice, where the imageswere taken at 0, 3, 6, 12, 24, and 48 hours after the exosomes wereintranasally administered to the mice, and where the exosomes wereisolated from EL4 cells:

FIG. 8A is a graph showing the concentration of curcumin in the braintissue of mice intranasally-administered a single dose of exosomalcurcumin;

FIG. 8B is a graph showing the concentration of curcumin in the brainsof mice intranasally-administered doses of exosomal curcumin every 12hours;

FIG. 9 includes images of brain sections of mice showing the presence ofPHK26-labeled EL4 exosomes at 15, 30, and 60 minutes after theintranasal administration of the exosomes (upper images) and images ofbrain sections of mice showing the presence of PHK26-labeled exosomes inmicroglia cells as evidenced by the co-localization of the PHK26-labeledexosomes with the anti-microglia cell marker IBa-1 (lower images);

FIG. 10A includes images of mice brains where the mice wereintraperitoneally injected with bacterial LPS (2.5. mg/kg) immediatelyprior to the intranasal administration of PBS, bovine serum albumin, EL4exosomes, free curcumin, or exosomal curcumin;

FIG. 10B includes graphs showing the FACS analysis of leukocytesisolated from the brains of mice two hours after the mice wereintraperitoneally injected with bacterial LPS (2.5 mg/kg) immediatelyprior to the intranasal administration of PBS, bovine serum albumin, EL4exosomes, free curcumin, or exosomal curcumin;

FIG. 10C includes graphs showing the percentage of CD45.2⁺IL-1β⁺ brainleukocytes and the quantitative real-time polymerase chain reaction(RT-PCR) analysis of IL-1β mRNA levels in mice intraperitoneallyinjected with bacterial LPS (2.5 mg/kg) immediately prior to theintranasal administration of PBS, bovine serum albumin, EL4 exosomes,free curcumin, or exosomal curcumin;

FIG. 10D includes terminal deoxynucleotidyl transferase dUTP nick endlabeling (TUNEL)-stained images of frozen brain sections of miceintraperitoneally injected with bacterial LPS (2.5 mg/kg) immediatelyprior to the intranasal administration of PBS, bovine serum albumin, EL4exosomes, free curcumin, or exosomal curcumin;

FIG. 10E includes images of brain leukocytes at 10× and 40×magnifications, where the brain leukocytes were isolated from mice 0.5,1, 2, and 6 hours after the mice were intraperitoneally injected withbacterial LPS (2.5 mg/kg) and were then immediatelyintranasally-administered exosomal curcumin, and where the brainleukocytes were TUNEL-stained, anti-IBa-1-stained, or co-stained usingboth TUNEL and anti-IBa-1 staining;

FIG. 10F includes images of FACS analysis of brain leukocytes from mice0.5, 2, and 6 hours after the mice were intraperitoneally injected withbacterial LPS (2.5 mg/kg) and were then immediatelyintranasally-administered exosomal curcumin, where the leukocytes werelabeled with Annexin-V to identify apoptotic cells;

FIG. 10G is a graph showing the percentages of Annexin V⁺ cells as afunction of the concentration of exosomal curcumin in the brain tissueof mice that were intraperitoneally injected with bacterial LPS (2.5mg/kg) immediately prior the intranasal administration of exosomalcurcumin;

FIG. 11A is a graph showing the clinical scores for experimentalautoimmune encephalitis (EAE) in mice that were administered a MyelinOligodendrocyte Glycoprotein (MOG) 35-55 peptide to induce EAE and werethen intranasally administered PBS, EL4 exosomes alone, curcumin, orexosomal curcumin starting 4 days after the administration of the MOGpeptide and continuing until the mice were sacrificed at day 35 post MOGadministration;

FIG. 11B is a graph showing the fold-change in IL-1β mRNA expressionlevels, as compared to a PBS control, in mice administered an MOG 35-55peptide to induce EAE and then intranasally administered PBS, EL4exosomes alone, curcumin, or exosomal curcumin;

FIG. 12A includes images and a graph showing the photon emissions ofmice injected with GL26 brain tumor cells and then intranasallyadministered PBS, EL4 exosomes alone, the Stat3 inhibitor JSI-124, orexosomal JSI-124;

FIG. 12B is a graph showing the percentage of surviving mice after themice were injected with GL26 brain tumor cells and then intranasallyadministered PBS, EL4 exosomes alone, the Stat3 inhibitor JSI-124, orexosomal JSI-124;

FIG. 12C is an image of a western blot for phospho-Stat3 in miceinjected with GL26 brain tumor cells and then intranasally administeredPBS, EL4 exosomes alone, the Stat3 inhibitor JSI-124, or exosomalJSI-124;

FIG. 12D is a graph showing RT-PCR analysis of IL-1β and IL-6 mRNAlevels in mice injected with GL26 brain tumor cells and thenintranasally administered PBS, EL4 exosomes alone, the Stat3 inhibitorJSI-124, or exosomal JSI-124.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

The details of one or more embodiments of the presently-disclosedsubject matter are set forth in this document. Modifications toembodiments described in this document, and other embodiments, will beevident to those of ordinary skill in the art after a study of theinformation provided in this document. The information provided in thisdocument, and particularly the specific details of the describedexemplary embodiments, is provided primarily for clearness ofunderstanding and no unnecessary limitations are to be understoodtherefrom. Additionally, while the following terms are believed to bewell understood by one of ordinary skill in the art, definitions are setforth to facilitate explanation of the presently-disclosed subjectmatter.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the presently-disclosed subject matter belongs.Although any methods, devices, and materials similar or equivalent tothose described herein can be used in the practice or testing of thepresently-disclosed subject matter, representative methods, devices, andmaterials are now described.

Following long-standing patent law convention, the terms “a,” “an,” and“the” refer to “one or more” when used in this application, includingthe claims. Thus, for example, reference to “a cell” includes aplurality of such cells, and so forth.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as reaction conditions, and so forth usedin the specification and claims are to be understood as being modifiedin all instances by the term “about.” Accordingly, unless indicated tothe contrary, the numerical parameters set forth in this specificationand claims are approximations that can vary depending upon the desiredproperties sought to be obtained presently-disclosed subject matter.

As used herein, the term “about,” when referring to a value or to anamount of mass, weight, time, volume, concentration or percentage ismeant to encompass variations of in some embodiments ±20%, in someembodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, insome embodiments ±0.5%, and in some embodiments ±0.1% from the specifiedamount, as such variations are appropriate to perform the disclosedmethod. As also used herein, ranges can be expressed as from “about” oneparticular value, and/or to “about” another particular value. It is alsounderstood that there are a number of values disclosed herein, and thateach value is also herein disclosed as “about” that particular value inaddition to the value itself. For example, if the value “10” isdisclosed, then “about 10” is also disclosed. It is also understood thateach unit between two particular units are also disclosed. For example,if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

Exosomes are naturally existing nanoparticles that are secretedendogenously by many types of in vitro cell cultures and in vivo cells,and are commonly found in vivo in body fluids, such as blood, urine andmalignant ascites. Exosomes are cup-like multivesicular bodies (MVBs)varying in size between 30-100 nm (12). MVBs are specialized endosomesin the endocytosis pathway of cells and are formed by inward budding andscission of vesicles from the limiting membranes into the endosomallumen (13). During the formation of MVBs, transmembrane and peripheralmembrane proteins are absorbed into the vesicle membrane, and at thesame time, cytosolic components are also embedded in the vesicles. Asthis process progresses, the MVBs ultimately fuse with the cellularmembrane, triggering the release of the exosomes from the cells.

During this process, unwanted molecules are eliminated from cells.However, cytosolic and plasma membrane proteins are also incorporatedduring this process into the exosomes, resulting in exosomes havingparticle size properties, lipid bilayer functional properties, and otherunique functional properties that allow the exosomes to potentiallyfunction as effective nanoparticle carriers of therapeutic agents. Inthis regard, it has now been discovered that exosomes can be used aspart of a specific nanoparticle-therapeutic agent delivery system thatis able to deliver a therapeutic agent to target cells and tissues,while also retaining the biological activity of the therapeutic agents.In particular, it has been observed that the formation ofexosome-therapeutic agent complexes results in an increase in thesolubility and stability of the therapeutic agents as well as anincrease in their bioavailability, all of which have been majorobstacles in the treatment of inflammatory disorders and cancers.

The presently-disclosed subject matter thus relates to exosomalcompositions that include therapeutic agents and are useful in thetreatment of various diseases, including inflammatory disorders andcancer. In some embodiments of the presently-disclosed subject matter,an exosomal composition is provided that comprises a therapeutic agentselected from a phytochemical agent, a chemotherapeutic agent, a Stat3inhibitor, or combinations thereof. In some embodiments, the therapeuticagent is encapsulated by an exosome to thereby provide an exosomalcomposition that displays increased in vitro and in vivo solubility,stability, and bioavailability as compared to the free (i.e.,non-encapsulated or unbound) therapeutic agent.

The phrase “encapsulated by an exosome,” or grammatical variationsthereof is used interchangeably herein with the phrase “exosomaltherapeutic agent” or “exosomal composition” to refer to exosomes whoselipid bilayer surrounds a therapeutic agent. For example, a reference to“exosomal curcumin” refers to an exosome whose lipid bilayerencapsulates or surrounds an effective amount of curcumin.

In some embodiments, the encapsulation of various therapeutic agentswithin exosomes can be achieved by first mixing the one or more of thephytochemical agents, Stat3 inhibitors, or chemotherapeutic agents withisolated exosomes in a suitable buffered solution, such asphosphate-buffered saline (PBS). After a period of incubation sufficientto allow the therapeutic agent to become encapsulated during theincubation period, the exosome/therapeutic agent mixture is thensubjected to a sucrose gradient (e.g., and 8, 30, 45, and 60% sucrosegradient) to separate the free therapeutic agent from the therapeuticagents encapsulated within the exosomes, and a centrifugation step toisolate the exosomes. After this centrifugation step, the exosomaltherapeutic agents are seen as a band in the sucrose gradient such thatthey can then be collected, washed, and dissolved in a suitable solutionfor use as described herein below.

As noted, in some embodiments, the therapeutic agent is a phytochemicalagent. As used herein, the term “phytochemical agent” refers to anon-nutritive plant-derived compound, or an analog thereof. Examples ofphytochemical agents include, but are not limited to compounds such asmonophenols; flavonoids, such as flavonols, flavanones, flavones,flavan-3-ols, anthocyanins, anthocyanidins, isoflavones,dihydroflavonols, chalcones, and coumestans; phenolic acids;hydroxycinnamic acids; lignans; tyrosol esters; stillbenoids;hydrolysable tannins; carotenoids, such as carotenes and xanthophylls;monoterpenes; saponins; lipids, such as phytosterols, tocopherols, andomega-3,6,9 fatty acids; diterpenes; triterpinoids; betalains, such asbetacyanins and betaxanthins; dithiolthiones; thiosulphonates; indoles;and glucosinolates. As another example of a phytochemical agentdisclosed herein, the phytochemical agent can be an analog of aplant-derived compound, such as oltipraz, which is an analog of1,2-dithiol-3-thione, a compound that is found in many calciferousvegetables.

In some embodiments of the presently-disclosed subject matter, thetherapeutic agent is a phytochemical agent selected from curcuminresveratrol, baicalein, equol, fisetin, and quercetin. In someembodiments, the phytochemical agent is curcumin. Curcumin is apleiotropic natural polyphenol with anti-inflammatory, anti-neoplastic,anti-oxidant and chemopreventive activity, with these activities havingbeen identified at both the protein and molecular levels (14, 15).Nevertheless, limited progress has been reported with respect to thetherapeutic use of curcumin as curcumin is insoluble in aqueous solventsand is relatively unstable. In addition, curcumin is known to have a lowsystemic bioavailability after oral dosing, which further limits itsusage and clinical efficacy. It has been determined, however, that byencapsulating curcumin in exosomes, not only can the solubility ofcurcumin be increased, but the encapsulation of the curcumin within theexosomes protects the curcumin from degradation and also increases thebioavailability of the exosornal curcumin.

As also noted herein above, in some embodiments of thepresently-disclosed subject matter, the therapeutic agent that isencapsulated within the exosome is a chemotherapeutic agent. Examples ofchemotherapeutic agents that can be used in accordance with thepresently-disclosed subject matter include, but are not limited to,platinum coordination compounds such as cisplatin, carboplatin oroxalyplatin; taxane compounds, such as paclitaxel or docetaxel;topoisomerase I inhibitors such as camptothecin compounds for exampleirinotecan or topotecan; topoisomerase II inhibitors such as anti-tumorpodophyllotoxin derivatives for example etoposide or teniposide;anti-tumor vinca alkaloids for example vinblastine, vincristine orvinorelbine; anti-tumor nucleoside derivatives for example5-fluorouracil, gemcitabine or capecitabine; alkylating agents, such asnitrogen mustard or nitrosourea for example cyclophosphamide,chlorambucil, carmustine or lomustine; anti-tumor anthracyclinederivatives for example daunorubicin, doxorubicin, idarubicin ormitoxantrone; HER2 antibodies for example trastuzumab; estrogen receptorantagonists or selective estrogen receptor modulators for exampletamoxifen, toremifene, droloxifene, faslodex or raloxifene; aromataseinhibitors, such as exemestane, anastrozole, letrazole and vorozole;differentiating agents such as retinoids, vitamin D and retinoic acidmetabolism blocking agents (RAMBA) for example accutane; DNA methyltransferase inhibitors for example azacytidine; kinase inhibitors forexample flavoperidol, imatinib mesylate or gefitinib;farnesyltransferase inhibitors; HDAC inhibitors; other inhibitors of theubiquitin-proteasome pathway for example VELCADE® (MillenniumPharmaceuticals, Cambridge, Mass.); or YONDELIS® (Johnson & Johnson, NewBrunswick, N.J.). In some embodiments, the chemotherapeutic agent thatis encapsulated by an exosome in accordance with the presently-disclosedsubject matter is selected from retinoic acid, 5-fluorouracil,vincristine, actinomycin D, adriamycin, cisplatin, docetaxel,doxorubicin, and taxol.

As further noted, in some embodiments, the therapeutic agent is a signaltransducer and activator of transcription 3 (Stat3) inhibitor. “Stat3”or “Signal Transducer and Activator of Transcription 3” is atranscription factor encoded by the STAT3 gene and, in response tocytokines or growth factors, is known to become phosphorylated and tothen translocate to the nucleus of cells where it mediates theexpression of a variety of genes in response to various stimuli, andthus plays a key role in a number of cellular processes including cellgrowth and apoptosis. In this regard, the term “Stat3 inhibitor” is usedherein to refer to any chemical compound or protein that prevents orotherwise reduces the activity of Stat3 including, but not limited to,chemical compounds or proteins that prevent or reduce thetranscriptional activity of Stat3, and chemical compounds or proteinsthat prevent or reduce the activation of Stat3 by preventing itsactivation (e.g., the phosphorylation and/or translocation of Stat3 tothe nucleus of a cell). A number of Stat3 inhibitors are known to thoseskilled in the art including, but not limited to, the PIAS3 protein,Stattin, or JSI-124, which is also referred to as curcurbitacin I. Insome embodiments of the presently-disclosed subject matter, the Stat3inhibitor that is encapsulated within the exosome is JSI-124.

The exosomes used to produce the exosomal compositions of thepresently-disclosed subject matter can be obtained from a variety ofsources using methods known to those of ordinary skill in the art. Theterm “isolated,” when used in the context of an exosome isolated from acell, refers to an exosome that, by the hand of man, exists apart fromits native environment and is therefore not a product of nature. Forexample, in some embodiments, the exosomes are isolated from the juicesof fruit (e.g., grape, grapefruit, and tomatoes). As another example, insome embodiments, the exosomes are isolated from cells by collectingcell culture supernatants and then purifying the exosomes from thesupernatants using known differential centrifugation techniques toisolate exosomes (see, e.g., Liu C, Yu S, Zinn K. et al. Murine mammarycarcinoma exosomes promote tumor growth by suppression of NK cellfunction. J. Immunol. 176(3), 1375-1385 (2006)). As such, in someembodiments, the exosomes that are used in accordance with thepresently-disclosed subject matter are isolated from a cell. In someembodiments, the cell is a cultured cell, that is, a cell propagated exvivo in culture media. In some embodiments, the culture cell can beimmortalized to facilitate continuous propagation. In some embodiments,the cell is a cancer cell, such as for example a cancer cell originallyisolated from a tumor and then propagated in culture, as is generallyknown in the art. In some embodiments, the cancer cell can be a lymphomacell, a breast cancer cell, or an adenocarcinoma cell.

In some embodiments of the presently-disclosed subject matter, theexosomal compositions of the presently-disclosed subject matterspecifically bind to a target cell or tissue. Applicants have discoveredthat exosomes released from different types of cells (i.e., derived fromdifferent cells) with different levels of activation (e.g. proliferatingvs. non-proliferating) exhibit tissue- and/or cell-specific in vivotropism, which can advantageously be utilized to direct the exosomes andthe exosomal compositions to a specific cell or tissue. For example, insome embodiments, the exosome used to produce an exosomal composition ofthe presently-disclosed subject matter is derived from a T lymphocyteand specifically binds CD11b⁺Gr1⁺ myeloid cells.

In some embodiments of the presently disclosed subject matter, apharmaceutical composition is provided that comprises an exosomalcomposition disclosed herein and a pharmaceutical vehicle, carrier, orexcipient. In some embodiments, the pharmaceutical composition ispharmaceutically-acceptable in humans. Also, as described further below,the pharmaceutical composition can be formulated as a therapeuticcomposition for delivery to a subject in some embodiments.

A pharmaceutical composition as described herein preferably comprises acomposition that includes pharmaceutical carrier such as aqueous andnon-aqueous sterile injection solutions that can contain antioxidants,buffers, bacteriostats, bactericidal antibiotics and solutes that renderthe formulation isotonic with the bodily fluids of the intendedrecipient, and aqueous and non-aqueous sterile suspensions, which caninclude suspending agents and thickening agents. The pharmaceuticalcompositions used can take such forms as suspensions, solutions oremulsions in oily or aqueous vehicles, and can contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.Additionally, the formulations can be presented in unit-dose ormulti-dose containers, for example sealed ampoules and vials, and can bestored in a frozen or freeze-dried or room temperature (lyophilized)condition requiring only the addition of sterile liquid carrierimmediately prior to use.

In some embodiments, solid formulations of the compositions for oraladministration can contain suitable carriers or excipients, such as cornstarch, gelatin, lactose, acacia, sucrose, microcrystalline cellulose,kaolin, mannitol, dicalcium phosphate, calcium carbonate, sodiumchloride, or alginic acid. Disintegrators that can be used include, butare not limited to, microcrystalline cellulose, corn starch, sodiumstarch glycolate, and alginic acid. Tablet binders that can be usedinclude acacia, methylcellulose, sodium carboxymethylcellulose,polyvinylpyrrolidone, hydroxypropyl methylcellulose, sucrose, starch,and ethylcellulose. Lubricants that can be used include magnesiumstearates, stearic acid, silicone fluid, talc, waxes, oils, andcolloidal silica. Further, the solid formulations can be uncoated orthey can be coated by known techniques to delay disintegration andabsorption in the gastrointestinal tract and thereby provide asustained/extended action over a longer period of time. For example,glyceryl monostearate or glyceryl distearate can be employed to providea sustained-/extended-release formulation. Numerous techniques forformulating sustained release preparations are known to those ofordinary skill in the art and can be used in accordance with the presentinvention, including the techniques described in the followingreferences: U.S. Pat. Nos. 4,891,223; 6,004,582; 5,397,574; 5,419,917;5,458,005; 5,458,887; 5,458,888; 5,472,708; 6,106,862; 6,103,263;6,099,862; 6,099,859; 6,096,340; 6,077,541; 5,916,595; 5,837,379;5,834,023; 5,885,616; 5,456,921; 5,603,956; 5,512,297; 5,399,362;5,399,359; 5,399,358; 5,725,883; 5,773,025; 6,110,498; 5,952,004;5,912,013; 5,897,876; 5,824,638; 5,464,633; 5,422,123; and 4,839,177;and WO 98/47491, each of which is incorporated herein by this reference.

Liquid preparations for oral administration can take the form of, forexample, solutions, syrups or suspensions, or they can be presented as adry product for constitution with water or other suitable vehicle beforeuse. Such liquid preparations can be prepared by conventional techniqueswith pharmaceutically acceptable additives such as suspending agents(e.g., sorbitol syrup, cellulose derivatives or hydrogenated ediblefats); emulsifying agents (e.g. lecithin or acacia); non-aqueousvehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionatedvegetable oils); and preservatives (e.g., methyl orpropyl-p-hydroxybenzoates or sorbic acid). The preparations can alsocontain buffer salts, flavoring, coloring and sweetening agents asappropriate. Preparations for oral administration can be suitablyformulated to give controlled release of the active compound. For buccaladministration the compositions can take the form of capsules, tabletsor lozenges formulated in conventional manner.

Various liquid and powder formulations can also be prepared byconventional methods for inhalation into the lungs of the subject to betreated or for intranasal administration into the nose and sinuscavities of a subject to be treated. For example, the compositions canbe conveniently delivered in the form of an aerosol spray presentationfrom pressurized packs or a nebulizer, with the use of a suitablepropellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas.Capsules and cartridges of, for example, gelatin for use in an inhaleror insufflator may be formulated containing a powder mix of the desiredcompound and a suitable powder base such as lactose or starch.

The compositions can also be formulated as a preparation forimplantation or injection. Thus, for example, the compositions can beformulated with suitable polymeric or hydrophobic materials (e.g., as anemulsion in an acceptable oil) or ion exchange resins, or as sparinglysoluble derivatives (e.g., as a sparingly soluble salt).

Injectable formulations of the compositions can contain various carrierssuch as vegetable oils, dimethylacetamide, dimethylformamide, ethyllactate, ethyl carbonate, isopropyl myristate, ethanol, polyols(glycerol, propylene glycol, liquid polyethylene glycol), and the like.For intravenous injections, water soluble versions of the compositionscan be administered by the drip method, whereby a formulation includinga pharmaceutical composition of the present invention and aphysiologically-acceptable excipient is infused.Physiologically-acceptable excipients can include, for example, 5%dextrose, 0.9% saline, Ringer's solution or other suitable excipients.Intramuscular preparations, e.g., a sterile formulation of a suitablesoluble salt form of the compounds, can be dissolved and administered ina pharmaceutical excipient such as Water-for-Injection, 0.9% saline, or5% glucose solution. A suitable insoluble form of the composition can beprepared and administered as a suspension in an aqueous base or apharmaceutically-acceptable oil base, such as an ester of a long chainfatty acid, (e.g., ethyl oleate).

In addition to the formulations described above, the exosomaltherapeutic agents of the present invention can also be formulated asrectal compositions, such as suppositories or retention enemas, e.g.,containing conventional suppository bases such as cocoa butter or otherglycerides. Further, the exosomal compositions can also be formulated asa depot preparation by combining the compositions with suitablepolymeric or hydrophobic materials (for example as an emulsion in anacceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salt.

Further provided, in some embodiments of the presently-disclosed subjectmatter, are methods for treating an inflammatory disorder. In someembodiments, a method for treating an inflammatory disorder is providedthat comprises administering to a subject in need thereof an effectiveamount of an exosomal composition of the presently-disclosed subjectmatter. In some embodiments of the presently-disclosed methods oftreating an inflammatory disorder, the therapeutic agent encapsulated byan exosome is a phytochemical agent and/or a Stat-3 inhibitor.

As used herein, the terms “treatment” or “treating” relate to anytreatment of a condition of interest (e.g., an inflammatory disorder ora cancer), including but not limited to prophylactic treatment andtherapeutic treatment. As such, the terms “treatment” or “treating”include, but are not limited to: preventing a condition of interest orthe development of a condition of interest; inhibiting the progressionof a condition of interest; arresting or preventing the furtherdevelopment of a condition of interest; reducing the severity of acondition of interest; ameliorating or relieving symptoms associatedwith a condition of interest; and causing a regression of a condition ofinterest or one or more of the symptoms associated with a condition ofinterest.

As used herein, the term “inflammatory disorder” includes diseases ordisorders which are caused, at least in part, or exacerbated, byinflammation, which is generally characterized by increased blood flow,edema, activation of immune cells (e.g., proliferation, cytokineproduction, or enhanced phagocytosis), heat, redness, swelling, painand/or loss of function in the affected tissue or organ. The cause ofinflammation can be due to physical damage, chemical substances,micro-organisms, tissue necrosis, cancer, or other agents or conditions.

Inflammatory disorders include acute inflammatory disorders, chronicinflammatory disorders, and recurrent inflammatory disorders. Acuteinflammatory disorders are generally of relatively short duration, andlast for from about a few minutes to about one to two days, althoughthey can last several weeks. Characteristics of acute inflammatorydisorders include increased blood flow, exudation of fluid and plasmaproteins (edema) and emigration of leukocytes, such as neutrophils.Chronic inflammatory disorders, generally, are of longer duration, e.g.,weeks to months to years or longer, and are associated histologicallywith the presence of lymphocytes and macrophages and with proliferationof blood vessels and connective tissue. Recurrent inflammatory disordersinclude disorders which recur after a period of time or which haveperiodic episodes. Some inflammatory disorders fall within one or morecategories. Exemplary inflammatory disorders include, but are notlimited to atherosclerosis; arthritis; inflammation-promoted cancers;asthma; autoimmune uveitis; adoptive immune response; dermatitis;multiple sclerosis; diabetic complications; osteoporosis; Alzheimer'sdisease; cerebral malaria; hemorrhagic fever; autoimmune disorders; andinflammatory bowel disease. In some embodiments, the inflammatorydisorder is an autoimmune disorder that, in some embodiments, isselected from lupus, rheumatoid arthritis, and autoimmuneencephalomyelitis.

In some embodiments, the inflammatory disorder is a brain-relatedinflammatory disorder. The term “brain-related inflammatory” disorder isused herein to refer to a subset of inflammatory disorders that arecaused, at least in part, or originate or are exacerbated, byinflammation in the brain of a subject. It has been determined that theexosomal compositions of the presently-disclosed subject matter areparticularly suitable for treating such disorders as those compositionsare able to cross the blood-brain barrier and effectively be used todeliver the therapeutic agents (e.g., curcumin or JSI-124) to the brainof a subject.

For administration of a therapeutic composition as disclosed herein(e.g., an exosome encapsulating a therapeutic agent), conventionalmethods of extrapolating human dosage based on doses administered to amurine animal model can be carried out using the conversion factor forconverting the mouse dosage to human dosage: Dose Human per kg=DoseMouse per kg×12 (Freireich, et al., (1966) Cancer Chemother Rep. 50:219-244). Drug doses can also be given in milligrams per square meter ofbody surface area because this method rather than body weight achieves agood correlation to certain metabolic and excretionary functions.Moreover, body surface area can be used as a common denominator for drugdosage in adults and children as well as in different animal species asdescribed by Freireich, et al, (Freireich et al., (1966) CancerChemother Rep. 50:219-244), Briefly, to express a mg/kg dose in anygiven species as the equivalent mg/sq m dose, multiply the dose by theappropriate km factor. In an adult human, 100 mg/kg is equivalent to 100mg/kg×37 kg/sq m=3700 mg/m².

Suitable methods for administering a therapeutic composition inaccordance with the methods of the presently-disclosed subject matterinclude, but are not limited to, systemic administration, parenteraladministration (including intravascular, intramuscular, and/orintraarterial administration), oral delivery, buccal delivery, rectaldelivery, subcutaneous administration, intraperitoneal administration,inhalation, intratracheal installation, surgical implantation,transdermal delivery, local injection, intranasal delivery, andhyper-velocity injection/bombardment. Where applicable, continuousinfusion can enhance drug accumulation at a target site (see, e.g., U.S.Pat. No. 6,180,082).

Regardless of the route of administration, the compositions of thepresently-disclosed subject matter are typically administered in amounteffective to achieve the desired response. As such, the term “effectiveamount” is used herein to refer to an amount of the therapeuticcomposition (e.g., an exosome encapsulating a therapeutic agent, and apharmaceutically vehicle, carrier, or excipient) sufficient to produce ameasurable biological response (e.g., a decrease in inflammation).Actual dosage levels of active ingredients in a therapeutic compositionof the present invention can be varied so as to administer an amount ofthe active compound(s) that is effective to achieve the desiredtherapeutic response for a particular subject and/or application. Ofcourse, the effective amount in any particular case will depend upon avariety of factors including the activity of the therapeuticcomposition, formulation, the route of administration, combination withother drugs or treatments, severity of the condition being treated, andthe physical condition and prior medical history of the subject beingtreated. Preferably, a minimal dose is administered, and the dose isescalated in the absence of dose-limiting toxicity to a minimallyeffective amount. Determination and adjustment of a therapeuticallyeffective dose, as well as evaluation of when and how to make suchadjustments, are known to those of ordinary skill in the art.

For additional guidance regarding formulation and dose, see U.S. Pat.Nos. 5,326,902; 5,234,933; PCT International Publication No. WO93/25521; Berkow et al., (1997) The Merck Manual of Medical Information,Home ed. Merck Research Laboratories, Whitehouse Station, N.J.; Goodmanet al., (1996) Goodman & Gilman's the Pharmacological Basis ofTherapeutics, 9th ed. McGraw-Hill Health Professions Division, New York;Ebadi, (1998) CRC Desk Reference of Clinical Pharmacology. CRC Press,Boca Raton, Fla.; Katzung, (2001) Basic & Clinical Pharmacology, 8th ed.Lange Medical Books/McGraw-Hill Medical Pub. Division, New York;Remington et al., (1975) Remington's Pharmaceutical Sciences, 15th ed.Mack Pub. Co., Easton, Pa.; and Speight et al., (1997) Avery's DrugTreatment: A Guide to the Properties, Choice, Therapeutic Use andEconomic Value of Drugs in Disease Management, 4th ed. AdisInternational, Auckland/Philadelphia; Duch et al., (1998) Toxicol. Lett.100-101:255-263.

In some embodiments of the therapeutic methods disclosed herein,administering an exosomal composition of the presently-disclosed subjectmatter reduces an amount of an inflammatory cytokine in a subject. Insome embodiments, the inflammatory cytokine can be interleukin-1β(IL-1β), tumor necrosis factor-alpha (TNE-α), or interleukin-6 (IL-6).

Various methods known to those skilled in the art can be used todetermine a reduction in the amount of inflammatory cytokines in asubject. For example, in certain embodiments, the amounts of expressionof an inflammatory cytokine in a subject can be determined by probingfor mRNA of the gene encoding the inflammatory cytokine in a biologicalsample obtained from the subject (e.g., a tissue sample, a urine sample,a saliva sample, a blood sample, a serum sample, a plasma sample, orsub-fractions thereof) using any RNA identification assay known to thoseskilled in the art. Briefly, RNA can be extracted from the sample,amplified, converted to cDNA, labeled, and allowed to hybridize withprobes of a known sequence, such as known RNA hybridization probesimmobilized on a substrate, e.g., array, or microarray, or quantitatedby real time PCR (e.g., quantitative real-time PCR, such as availablefrom Bio-Rad Laboratories, Hercules, Calif.). Because the probes towhich the nucleic acid molecules of the sample are bound are known, themolecules in the sample can be identified. In this regard, DNA probesfor one or more of the mRNAs encoded by the inflammatory genes can beimmobilized on a substrate and provided for use in practicing a methodin accordance with the presently-disclosed subject matter.

With further regard to determining levels of inflammatory cytokines insamples, mass spectrometry and/or immunoassay devices and methods canalso be used to measure the inflammatory cytokines in samples, althoughother methods can also be used and are well known to those skilled inthe art. See, e.g., U.S. Pat. Nos. 6,143,576; 6,113,855; 6,019,944;5,985,579; 5,947,124; 5,939,272; 5,922,615; 5,885,527; 5,851,776;5,824,799; 5,679,526; 5,525,524; and 5,480,792, each of which is herebyincorporated by reference in its entirety. Immunoassay devices andmethods can utilize labeled molecules in various sandwich, competitive,or non-competitive assay formats, to generate a signal that is relatedto the presence or amount of an analyte of interest. Additionally,certain methods and devices, such as biosensors and opticalimmunoassays, can be employed to determine the presence or amount ofanalytes without the need for a labeled molecule. See, e.g., U.S. Pat.Nos. 5,631,171; and 5,955,377, each of which is hereby incorporated byreference in its entirety.

Any suitable immunoassay can be utilized, for example, enzyme-linkedimmunoassays (ELISA), radioimmunoassays (RIAs), competitive bindingassays, and the like. Specific immunological binding of the antibody tothe inflammatory molecule can be detected directly or indirectly. Directlabels include fluorescent or luminescent tags, metals, dyes,radionucleotides, and the like, attached to the antibody. Indirectlabels include various enzymes well known in the art, such as alkalinephosphatase, horseradish peroxidase and the like.

The use of immobilized antibodies or fragments thereof specific for theinflammatory molecules is also contemplated by the present invention.The antibodies can be immobilized onto a variety of solid supports, suchas magnetic or chromatographic matrix particles, the surface of an assayplate (such as microtiter wells), pieces of a solid substrate material(such as plastic, nylon, paper), and the like. An assay strip can beprepared by coating the antibody or a plurality of antibodies in anarray on a solid support. This strip can then be dipped into the testbiological sample and then processed quickly through washes anddetection steps to generate a measurable signal, such as for example acolored spot.

Mass spectrometry (MS) analysis can be used, either alone or incombination with other methods (e.g., immunoassays), to determine thepresence and/or quantity of an inflammatory molecule in a subject.Exemplary MS analyses that can be used in accordance with the presentinvention include, but are not limited to: liquid chromatography-massspectrometry (LC-MS); matrix-assisted laser desorption/ionizationtime-of-flight MS analysis (MALDI-TOF-MS), such as for exampledirect-spot MALDI-TOF or liquid chromatography MALDI-TOF massspectrometry analysis; electrospray ionization MS (ESI-MS), such as forexample liquid chromatography (LC) ESI-MS; and surface enhanced laserdesorption/ionization time-of-flight mass spectrometry analysis(SELDI-TOF-MS). Each of these types of MS analysis can be accomplishedusing commercially-available spectrometers, such as, for example, triplequadropole mass spectrometers. Methods for utilizing MS analysis todetect the presence and quantity of peptides, such as inflammatorycytokines, in biological samples are known in the art. See, e.g., U.S.Pat. Nos. 6,925,389; 6,989,100; and 6,890,763 for further guidance, eachof which are incorporated herein by this reference.

With still further regard to the various therapeutic methods describedherein, although certain embodiments of the methods disclosed hereinonly call for a qualitative assessment (e.g., the presence or absence ofthe expression of an inflammatory cytokine in a subject), otherembodiments of the methods call for a quantitative assessment (e.g., anamount of increase in the level of an inflammatory cytokine in asubject). Such quantitative assessments can be made, for example, usingone of the above mentioned methods, as will be understood by thoseskilled in the art.

The skilled artisan will also understand that measuring a reduction inthe amount of a certain feature (e.g., cytokine levels) or animprovement in a certain feature (e.g., inflammation) in a subject is astatistical analysis. For example, a reduction in an amount ofinflammatory cytokines in a subject can be compared to control level ofinflammatory cytokines, and an amount of inflammatory cytokines of lessthan or equal to the control level can be indicative of a reduction inthe amount of inflammatory cytokines, as evidenced by a level ofstatistical significance. Statistical significance is often determinedby comparing two or more populations, and determining a confidenceinterval and/or a p value. See, e.g., Dowdy and Wearden, Statistics forResearch, John Wiley & Sons, New York, 1983, incorporated herein byreference in its entirety. Preferred confidence intervals of the presentsubject matter are 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9% and 99.99%,while preferred p values are 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001,and 0.0001.

Further provided, in some embodiments, are methods for treating acancer. In some embodiments, a method for treating a cancer is providedthat comprises administering to a subject in need thereof an effectiveamount of an exosomal composition of the presently-disclosed subjectmatter (i.e., where an exosome encapsulates a therapeutic agent). Insome embodiments, the therapeutic agent encapsulated within the exosomeand used to treat the cancer is selected from a phytochemical agent, achemotherapeutic agent, and a Stat3 inhibitor as such agents have beenfound to be particularly useful in the treatment of cancer. As usedherein, the term “cancer” refers to all types of cancer or neoplasm ormalignant tumors found in animals, including leukemias, carcinomas,melanoma, and sarcomas.

By “leukemia” is meant broadly progressive, malignant diseases of theblood-forming organs and is generally characterized by a distortedproliferation and development of leukocytes and their precursors in theblood and bone marrow. Leukemia diseases include, for example, acutenonlymphocytic leukemia, chronic lymphocytic leukemia, acutegranulocytic leukemia, chronic granulocytic leukemia, acutepromyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, aleukocythemic leukemia, basophylic leukemia, blast cell leukemia, bovineleukemia, chronic myelocytic leukemia, leukemia cutis, embryonalleukemia, eosinophilic leukemia, Gross' leukemia, hairy-cell leukemia,hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia,stem cell leukemia, acute monocytic leukemia, leukopenic leukemia,lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia,lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia,mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia,monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloidgranulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasmacell leukemia, plasmacytic leukemia, promyelocytic leukemia, Rieder cellleukemia, Schilling's leukemia, stem cell leukemia, subleukemicleukemia, and undifferentiated cell leukemia.

The term “carcinoma” refers to a malignant new growth made up ofepithelial cells tending to infiltrate the surrounding tissues and giverise to metastases. Exemplary carcinomas include, for example, acinarcarcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cysticcarcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolarcarcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinomabasocellulare, basaloid carcinoma, basosquamous cell carcinoma,bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogeniccarcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorioniccarcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma,cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum,cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma,carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiennoidcarcinoma, carcinoma epitheliale adenoides, exophytic carcinoma,carcinoma ex ulcere, carcinoma fibrosum, gelatiniform carcinoma,gelatinous carcinoma, giant cell carcinoma, carcinoma gigantocellulare,glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma,hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma,hyaline carcinoma, hypemephroid carcinoma, infantile embryonalcarcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelialcarcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma, large-cellcarcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatouscarcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullarycarcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma,carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma,carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes,nasopharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans,osteoid carcinoma, papillary carcinoma, periportal carcinoma,preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma,renal cell carcinoma of kidney, reserve cell carcinoma, carcinomasarcomatodes, schneiderian carcinoma, scirrhous carcinoma, carcinomascroti, signet-ring cell carcinoma, carcinoma simplex, small-cellcarcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cellcarcinoma, carcinoma spongiosum, squamous carcinoma, squamous cellcarcinoma, string carcinoma, carcinoma telangiectaticum, carcinomatelangiectodes, transitional cell carcinoma, carcinoma tuberosum,tuberous carcinoma, verrucous carcinoma, and carcinoma villosum.

The term “sarcoma” generally refers to a tumor which is made up of asubstance like the embryonic connective tissue and is generally composedof closely packed cells embedded in a fibrillar or homogeneoussubstance. Sarcomas include, for example, chondrosarcoma, fibrosarcoma,lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemethy'ssarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma,ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, choriocarcinoma, embryonal sarcoma, Wilns' tumor sarcoma, endometrial sarcoma,stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma,giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathicmultiple pigmented hemorrhagic sarcoma, immunoblastic sarcoma of Bcells, lymphoma, immunoblastic sarcoma of T-cells, Jensen's sarcoma,Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma,malignant mesenchymoma sarcoma, parosteal sarcoma, reticulocyticsarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, andtelangiectaltic sarcoma.

The term “melanoma” is taken to mean a tumor arising from themelanocytic system of the skin and other organs. Melanomas include, forexample, acral-lentiginous melanoma, amelanotic melanoma, benignjuvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passeymelanoma, juvenile melanoma, lentigo maligna melanoma, malignantmelanoma, nodular melanoma subungal melanoma, and superficial spreadingmelanoma.

Additional cancers include, for example, Hodgkin's Disease,Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, breast cancer,ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis,primary macroglobulinemia, small-cell lung tumors, primary brain tumors,stomach cancer, colon cancer, malignant pancreatic insulanoma, malignantcarcinoid, premalignant skin lesions, testicular cancer, lymphomas,thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tractcancer, malignant hypercalcemia, cervical cancer, endometrial cancer,and adrenal cortical cancer. In some embodiments, the cancer is selectedfrom the group consisting of skin cancer, head and neck cancer, coloncancer, breast cancer, brain cancer, and lung cancer.

In some particular embodiments, the cancer is a brain cancer such as, insome embodiments, a glioma. It has been discovered that administering anexosomal composition of the presently-disclosed subject matterintranasally to a subject results in the preferential targeting ofmicroglia cells by the exosomal compositions. Microglia cells are knownto play a role in many brain diseases and, more specifically, have beenimplicated in the progression of brain tumor growth and autoimmunediseases. The administration of a exosomal composition of thepresently-disclosed subject matter, however, results in a significantdecrease in the number of microglia cells in the brains of a subject anda concomitant reduction in brain tumor growth. In some embodiments ofthe methods of treating a cancer disclosed herein, the exosome isadministered to the subject by an intranasal, oral, or intratumoralroute of administration to thereby treat the cancer. In someembodiments, the exosome specifically binds to a cancerous cell ortissue to thereby precisely deliver the therapeutic agent to theaffected cell or tissue of the subject.

As used herein, the term “subject” includes both human and animalsubjects. Thus, veterinary therapeutic uses are provided in accordancewith the presently disclosed subject matter. As such, thepresently-disclosed subject matter provides for the treatment of mammalssuch as humans, as well as those mammals of importance due to beingendangered, such as Siberian tigers; of economic importance, such asanimals raised on farms for consumption by humans; and/or animals ofsocial importance to humans, such as animals kept as pets or in zoos.Examples of such animals include but are not limited to: carnivores suchas cats and dogs; swine, including pigs, hogs, and wild boars; ruminantsand/or ungulates such as cattle, oxen, sheep, giraffes, deer, goats,bison, and camels; and horses. Also provided is the treatment of birds,including the treatment of those kinds of birds that are endangeredand/or kept in zoos, as well as fowl, and more particularly domesticatedfowl, i.e., poultry, such as turkeys, chickens, ducks, geese, guineafowl, and the like, as they are also of economic importance to humans.Thus, also provided is the treatment of livestock, including, but notlimited to, domesticated swine, ruminants, ungulates, horses (includingrace horses), poultry, and the like.

The practice of the presently-disclosed subject matter can employ,unless otherwise indicated, conventional techniques of cell biology,cell culture, molecular biology, transgenic biology, microbiology,recombinant DNA, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature. See e.g.,Molecular Cloning A Laboratory Manual (1989), 2nd Ed., ed. by Sambrook,Fritsch and Maniatis, eds., Cold Spring Harbor Laboratory Press,Chapters 16 and 17; U.S. Pat. No. 4,683,195; DNA Cloning, Volumes I andII, Glover, ed., 1985; Oligonucleotide Synthesis, M. J. Gait, ed., 1984;Nucleic Acid Hybridization, D. Hames & S. J. Higgins, eds., 1984;Transcription and Translation, B. D. Hames & S. J. Higgins, eds., 1984;Culture Of Animal Cells, R. I. Freshney, Alan R. Liss, Inc., 1987;Immobilized Cells And Enzymes, IRL Press, 1986; Perbal (1984), APractical Guide To Molecular Cloning; See Methods In Enzymology(Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells,J. H. Miller and M. P. Calos, eds., Cold. Spring Harbor Laboratory,1987; Methods In Enzymology, Vols. 154 and 155, Wu et al., eds.,Academic Press Inc., N.Y.; Immunochemical Methods In Cell And MolecularBiology (Mayer and Walker, eds., Academic Press, London, 1987; HandbookOf Experimental Immunology, Volumes I-IV, D. M. Weir and C. C.Blackwell, eds., 1986.

The presently-disclosed subject matter is further illustrated by thefollowing specific but non-limiting examples. Some of the followingexamples are prophetic, notwithstanding the numerical values, resultsand/or data referred to and contained in the examples. The followingexamples may also include compilations of data that are representativeof data gathered at various times during the course of development andexperimentation related to the present invention.

EXAMPLES Materials and Methods for Examples 1-4

Mice.

7- to 10-week female C57BL/6j mice (The Jackson Laboratory, Bar Harbor,Me.) were used. All animal studies were conducted within the guidelinesestablished by an Institutional Animal Care and Use Committee.

Chemicals and Reagents.

Curcumin and lipopolysaccharide (LYS) were purchased from Sigma-AldrichCo.

Cell Culture.

An EL-4 mouse lymphoma cell line and RAW 264.7 murine macrophage cellline were utilized and were maintained in vitro at 37° C. in ahumidified 5% CO₂ atmosphere in air with complete RPMI1640 and DMEMmedium supplemented with 10% fetal bovine serum (FBS). The FBS that wasused in cell cultures to isolate exosomes was exosome-depleted bydifferential centrifugation using a method described previously (22).

Preparation of Exosomes.

The cell culture supernatants were collected and used for exosomepurification by differential centrifugation using a previously describedmethod (22). Purity and integrity of sucrose gradient isolated exosomeswere analyzed using a Hitachi H7000 electron microscope as previouslydescribed (23). The concentration of exosomes was determined byanalyzing protein concentration using a standard protein quantitationassay kit with bovine serum albumin (BSA) as a standard. The proteinexpression of exosomes was determined by western blotting analysis asdescribed previously (23).

Preparation of Exosomal Curcumin.

Exosomal curcumin was prepared by mixing curcumin with EL4 exosomes inPBS. After incubation at 22° C. for 5 min, the mixture was subjected tosucrose gradient (8%, 30%, 45% and 60%, respectively) centrifugation for1.5 h at 36,000 rpm. The exosomal curcumin, distinguished as a yellowishband in the sucrose gradient between 45% and 60%, was subsequentlycollected, washed and dissolved with PBS. The concentration of exosomesand curcumin within the complex was then determined as described below.Based on the morphology and protein expression, the vesicles werefurther determined to be exosomes (23).

Analysis of Curcumin Concentration In Vitro and In Vivo.

The concentration of curcumin in samples in vitro was determined using aNanodrop 1000 spectrophotometer (NanoDrop product, Wilmington, Del.) at420 nm. Briefly, to evaluate the concentration of curcumin, a standardcurve of curcumin was plotted first. A stock solution of curcumin wasdiluted to a range of 5 to 50 μM. A standard calibration curve wasobtained by plotting the concentration of standard curcumin versusfluorescent absorbance at 420 nm (OD₄₂₀). The curcumin quantity in cellculture supernatant or PBS was calculated based on the OD₄₂₀ withrespect to the concentration of curcumin in the diluted standards.

High-performance liquid chromatography (HPLC) analysis was then adaptedto determine the concentration of curcumin as described previously (24).To determine the concentration of curcumin in plasma, a standard curveof curcumin was plotted first. Briefly, a stock solution of curcumin(0.5 mg/ml) in acetonitrile was diluted to a range of 0.1 to 5 μg/mlwith acetonitrile, and then 10 μl of diluted curcumin were added to 90μl of plasma isolated from naïve C57BL/6j mice (a range of 1-500 ng/ml).The mixture was added to an equal volume of emodin (0.15 μg/ml, Sigma,St. Louis, Mo.) and vortexed for 5 min at 22° C. After centrifugation at2500×g for 15 minutes to remove precipitated plasma proteins, 50 μl ofeach of the working solutions containing 1 to 500 ng/ml of curcumin wasthen analyzed by HPLC. The chromatographic separation was performed on aC18 column (aappTec, 5 μm, 250×4.6 mm) with the mobile phase composed ofacetonitrile-5% acetic acid (75:25, v/v) at a flow rate of 1.0 ml/min.The wavelength of detection was at 420 nm. A standard calibration curvewas obtained by plotting the concentration of standard curcumin versusabsorbance units (AU).

To determine the concentration of curcumin in the samples, plasmasamples collected from mice treated with exosomal curcumin or freecurcumin were precipitated with emodin to remove proteins and analyzedusing an identical method as described above. The concentration wascalculated using the absorbance units with respect to the concentrationof curcumin in the standard curve.

FACS Analysis.

For cell surface marker staining, isolated cells were blocked at 4° C.for 5 min with 10 μg/ml mouse Fc block (BD Biosciences, San Jose,Calif.) and then reacted with various fluorochrome-labeled antibodiesincluding appropriate isotype controls for 30 min at 4° C. After washingtwice, cells were fixed and analyzed using a FACSCalibur flow cytometer(Becton Dickinson Biosciences, San Jose, Calif.). Data were analyzedusing FlowJo software (Tree Star, Inc., Ashland, Oreg.). The followingantibodies were used for immunostaining: FITC-Annexing V (Invitrogen,Carlsbad, Calif.), APC anti-mouse CD11b and PE anti-mouse Gr-1(eBiosciences, San Diego, Calif.).

In Vitro Stability Assays.

To determine the stability of free curcumin and exosomal curcumin in PBS(pH 7.4), curcumin and exosomal curcumin were added to 2 ml PBS toachieve a final concentration of 30 μM and incubated in the dark in a37° C.′ water bath. At different time points, 100 μL of each sample weretaken to determine the concentration of curcumin. The concentrations ofcurcumin or exosomal curcumin at the beginning were considered as 1.00.The fold reduction of the concentration at each time was determined bycomparison to the beginning value. The experiments were repeated threetimes for each time point (n=3).

In Vivo Bioavailability Assays.

To determine the bioavailability of free curcumin and exosomal curcuminin vivo, two groups (5 per group) of C57BL/6j mice wereintra-peritoneally (IP) injected or administrated orally with 100 mgcurcumin or exosomal curcumin/kg body weight. At 0.5 hr, 1 hr and 2 hr,blood samples were taken through eye bleeding and the concentration ofcurcumin in the plasma was determined by HPLC as described above. Naïvemice without treatment were used as blank controls.

In Vitro Pro-Inflammatory Cytokine Induction Assays.

RAW cells were plated on 24-well plates and incubated overnight. Thecells were treated with curcumin or exosomal curcumin at a concentrationof 20 μM for 1 h and then stimulated with lipopolysaccharide (LPS; 50ng/ml) for an additional 6 hr. RAW 264.7 cells treated with PBS orexosomes served as controls. Tumor necrosis factor-alpha (TNF-α□□□ andInterleukin-6 (IL-6) levels in the cell culture supernatant weremeasured using a standard ELISA (eBiosciences, San Diego, Calif.).

LPS Mouse Septic Shock Model.

Curcumin or exosomal curcumin (4 mg/kg of body weight) was IP injectedinto C57BL/6j mice together with LPS (18.5 mg/kg, Sigma, St. Louis,Mo.). EL-4 exosomes equal to the amount in exosomal curcumin and PBSwere used as controls. Mouse mortality was monitored over a period of 4days. The sera were collected 16 hr after LPS injection and used todetermine IL-6 and TNF-α levels using ELISAs as before. At day 1 afterLPS challenge, 3 mice from each treated group were sacrificed and theleukocytes in the lungs were isolated using a method describedpreviously (21). The percentage of CD11b⁺Gr-1⁺ cells in the lung wasdetermined by FACS analysis.

Isolation of Gr1⁺ Cells from Mouse Bone Marrow Cells.

Mouse bone marrow cells were isolated as described previously (22). Theisolated bone marrow cells were re-suspended to a concentration of 1×10⁸cells/ml using RPMI 1640 medium supplemented with 10% FBS. A Gr1-PEconjugated antibody (3 μg/ml), following a mouse FcR blocking specificantibody (5 μl/ml), was added, mixed thoroughly and incubated at 4° C.for 15 min. After centrifugation at 1500 rpm for 5 min, cells werere-suspended in fresh medium to a concentration of 1×10⁸ cells/ml.EasySep® PE selection cocktail (25 μl/ml, StemCell Technologies,Vancouver, British Columbia) was added to the cells and incubated at 4°C. for 15 min. Afterwards, EasySep® magnetic nanoparticles (25 μl/ml)were added and incubated at 4° C. for another 15 min. Culture medium wasadded to a final volume of 2.5 ml and the cells mixed by gentlepipetting of the mixture 2-3 times. The uncapped polystyrene tube wasplaced into the EasySep® magnet and set aside for 5 min. The supernatantcontaining unbound cells was removed leaving the magnetically bound Gr1⁺cells. A second round of magnetic separation was done on thesupernatant. Positively selected (magnetically bound) cells werecollected from the tubes, counted and cultured in RPMI 1640 supplementedwith MCSF (20 ng/ml) for curcumin uptake and apoptosis assays.

Statistical Analysis.

Statistical differences between groups were determined by ANOVA withmultiple comparisons using Fisher's post hoc analysis. The Student's ttest was used for comparisons when only two parameters were evaluated.P<0.05 was considered significant.

Example 1—Encapsulation of Curcumin into Exosomes

Exosomes are 30-100 nm nanoparticles (FIG. 1B) secreted by cells intothe extracellular environment. To determine if nanoparticle exosomescould be used as a carrier to entrap curcumin, curcumin was mixed withEL4-derived exosomes at 22° C., and then subjected to sucrose gradientcentrifugation. A yellowish band (FIG. 1A, right) appeared between the45% and 60% sucrose gradients, and a weak band appeared between the 30%and 45% gradients (original exosomes, FIG. 1A, left). The yellowish bandwas collected, washed and dissolved in PBS. This fraction was designatedexosomal curcumin. The morphology and the size of exosomal curcumin weresimilar to the original exosomes (FIG. 1B). Exosomal protein markers,such as TSG101 and CD81 (FIG. 1C) were identified in the exosomalcurcumin. The binding affinity was calculated to be approximately 2.9 gcurcumin to 1 g exosomes. Exosomes isolated from other types of celllines, including MDA-MB231 (human adenocarcinoma), 4T-1 (murine breasttumor cell line) and primary mouse embryonic fibroblasts, were alsoobserved to have also have similar efficiency in terms of bindingcurcumin.

Example 2—Encapsulation of Curcumin Into Exosomes Increases Curcumin'SSolubility, Stability and Bioavailability

Curcumin is a hydrophobic polyphenol compound that is insoluble inaqueous solution. To determine if the binding of curcumin to exosomesincreased the solubility of curcumin, an identical amount of curcuminwas mixed in an equal volume of PBS or exosomes in PBS, and the mixtureswere placed on ice for 30 min. To estimate curcumin solubility, theconcentration of curcumin in the supernatant was determined using aNanodrop 1000 spectrophotometer. The curcumin concentration in themixture of curcumin and exosomes was appropriately 5 fold higher thancurcumin alone (FIGS. 2A-2B). Thus, the solubility of exosomal curcuminis higher than free curcumin and this is believed to be due to thebinding of curcumin to exosomes.

Curcumin is relatively unstable, and this is one of the major barriersfor clinical use of curcumin to treat cancer and other inflammationrelated diseases (15). To determine whether exosomal curcumin is morestable, free curcumin and exosomal curcumin were incubated at 37° C.over a period of 150 min and sampled periodically to determine theconcentration of curcumin. After incubation for 150 min at 37° C., itwas found that free curcumin in PBS degraded quickly and only 25%remained after 150 min of incubation when compared to the 0 min sample(set as 1.0). Curcumin in exosomal curcumin was protected fromdegradation with more than 80% remaining after 150 min incubation in PBS(pH 7.4, FIG. 2C).

Another major barrier for clinical use of curcumin is its low systemicbioavailability (15). After administering curcumin orally, curcumin isdigested in the stomach. Along with other food, curcumin then passesthrough the intestinal wall into the enterohepatic circulation, arrivingin the liver for detoxification (the first pass effect) and eventuallygets into the blood stream. The low bioavailability of curcumin may bedue to the rapid first pass effect and the fast intestinalglucuronidation metabolism. Nanoparticles for drug delivery can increasedrug bioavailability through accumulating in the reticulo-endothelialsystem (RES) and achieving enhanced permeability and retention effects(EPR effect) (1). To assess if exosomal curcumin can increase thebioavailability of curcumin, free curcumin and exosomal curcumin (beforepurification, see Material & Methods for Example 1-4) was administeredIP or orally at a dose of 100 mg/kg of body weight. Due to the lowbioavailability of curcumin, a high dose of curcumin was used to achievedetectable curcumin in curcumin treated mice. Curcumin in the plasma wasthen quantified at 0.5, 1, and 2 hr after the administration using anestablished HPLC method (see, e.g., FIG. 3B). FIG. 3A shows that at 30min, IP administration of exosomal curcumin led to a 5 to 10 fold highercurcumin accumulated in peripheral blood than that of curcumin alone. At120 min after IP injection, curcumin in the plasma still remained at amuch higher level in the group of mice injected with exosomal curcumin.In contrast, there was no detectable curcumin circulating in the bloodof mice treated with curcumin alone. Similar results were obtained whenmice were administrated curcumin or exosomal curcumin orally.

Example 3—Anti-Inflammation Activity of Exosomal Curcumin

The foregoing data show that, as a nanoparticle drug carrier, exosomescan increase the solubility and stability of curcumin in vitro, and thebioavailability of curcumin in vivo. In this regard, it was hypothesizedthat exosomal curcumin can enhance the anti-inflammatory activity ofcurcumin through accumulating curcumin to a high level in cellulartargets. To evaluate the anti-inflammatory activity of exosomal curcuminin vitro, RAW 264.7 cells were treated with curcumin or exosomalcurcumin at a concentration of 20 μM for 1 hr. Subsequently treatedcells were stimulated with LPS (50 ng/ml) for an additional 6 hr.Cytokine production in the supernatant was measured 6 hr post treatment.As shown in FIG. 4, exosomal curcumin treated macrophages producedsignificantly less IL-6 and TNF-α in comparison with curcumin treatmentalone.

To assess the anti-inflammatory activity of exosomal curcumin in vivo, aLPS induced septic shock model was adapted. Briefly, to monitor micemortality, a LD₅₀ (median lethal dose) of LPS was determined first. LPS(5 mg/ml, Sigma, St. Louis, Mo.) was then prepared with sterile H₂O andvaried amounts of LPS were IP injected into the same batch ofcommercially supplied C57BL/6j mice. Each group contained 6 mice. TheLD₅₀ was 18.75 mg of LPS/kg of body weight. C57BL/6j mice were IPinjected with LPS (18.75 mg/kg) together with curcumin or exosomalcurcumin (4 mg/kg body weight) treatments. There was a significantsurvival advantage for mice treated with exosomal curcumin as comparedto mice treated with an equivalent concentration of free curcumin over a4-day period (FIG. 5A). Exosomes and PBS injections served as controls.Sixteen hours after IP injection of LPS, the sera levels of IL-6 andTNF-α were similar in mice treated with free curcumin, exosomes, andPBS. However, both cytokines were significantly lower in the exosomalcurcumin treated group of mice (FIG. 5B) and this finding correlatedwith mice mortality.

Example 4—Exosomal Curcumin Decreased CD11b⁺Gr1⁺ Cells in the Lungs ofMice

One of the features of LPS induced septic shock is a robust increase ofthe number of CD11b⁺Gr-1⁺ cells that are sequestered in the lungsleading to acute lung inflammation (26, 27). In LPS treated mice, therewere significantly fewer CD11b⁺Gr-1⁺ cells in the lungs, but not inother organs of the mice treated with exosomal curcumin when compared tomice treated with curcumin, exosomes or PBS (FIG. 6A). It has beenreported previously that tumor exosomes are taken up by CD11b⁺Gr-1⁺cells circulating in the peripheral blood (25, 28). To evaluate whetherexosomal curcumin can take advantage of this property, Gr1⁺ cells wereisolated from bone marrow cells and co-cultured with curcumin orexosomal curcumin at the same concentration. At 1, 2, and 3 hours,cellular curcumin concentration was significantly higher in exosomalcurcumin treated cells than free curcumin treated cells (FIG. 6B); anincrease in curcumin did not occur in cells treated with an equivalentamount of exosomes and curcumin together. Interestingly, curcumininduces Gr1⁺ cell apoptosis when Gr1⁺ cells are treated with 5 μM and 10μM curcumin, and exosomal curcumin can significantly enhance thisinduction as determined by FACS analysis (FIG. 6C). Gr1⁺ cells treatedwith an equivalent amount of exosomes and curcumin had a similarapoptosis induction as curcumin treatment alone, and Gr1⁺ cells treatedwith exosomes alone had a similar apoptosis percentage as the controls.Without wishing to be bound by any particular theory, it was thoughtthat the increase in curcumin uptake by Gr1⁺ cells with an increasedcell apoptosis caused by exosomal curcumin treatment can be the reasonfewer CD11b⁺Gr1⁺ cells were found in the lungs and the mice wereprotected from LPS induced septic death.

Discussion of Examples 1-4

The foregoing examples indicate that nanoparticle exosomes can carry anddeliver curcumin in a manner that enhances the anti-inflammatoryactivity of curcumin through: 1) increasing the solubility, stabilityand bioavailability of curcumin, and 2) enhancing and increasing thedelivery of curcumin to activated monocytes. The approach describedherein above also leads to the protection of mice from LPS inducedseptic shock. Furthermore, the above data demonstrates that exosomestarget not only CD11b⁺Gr-1⁺ cells in peripheral blood but also enhanceand increase the delivery of exosomal curcumin to CD11b⁺Gr-1⁺ cells,thus inducing more cell death. CD11b⁺Gr-1⁺ cells are one of the majorcellular populations associated with disease pathogenesis. Accumulationof CD11b⁺Gr-1⁺ cells can suppress host immune responses and interruptimmunosurveillance, which provides an explanation as to why long-terminflammation promotes tumor progression. Exosome directed curcumintargeting to CD11b⁺Gr-1⁺ cells provides a means to treat inflammatorydisorders and cancers.

As noted, the foregoing data indicate that encapsulation of curcumininto exosomes can increase the solubility, stability and bioavailabilityof curcumin. Exosomes, which contain a lipid bilayer, can load curcuminthrough physical entrapment. Through the hydrophobic interaction betweenthe hydrophobic tails and hydrophobic drug, curcumin can beself-assembled into the lipid bilayer of exosomes and this may protectcurcumin from degradation. Nanoparticles are distributed to organs in asize dependent selective manner (30). Sizes less than 5 nm nanoparticlesare preferentially distributed to kidney and liver. Larger sizedexosomes likely stay in the vasculature for an extended time. Whenformulated with curcumin, exosomes increase curcumin water solubilityand stability, and result in better delivery to the blood stream andincreased bioavailability.

Exosome-based drug delivery renders hydrophobic drugs, like curcumin,capable of being dispersed in aqueous environments, thus circumventingthe pitfalls of poor stability and bioavailability. The foregoingresults indicate that this strategy is applicable for treatingmonocyte-mediated acute inflammation-related diseases and can be usefulin preventing chronic inflammation triggered diseases, such as obesity,and cancers. Finally, unlike other non-host delivery vehicles,host-derived exosomes have an advantage as a potential delivery vehiclebecause they do not induce an immune response with subsequent sideeffects.

Materials and Methods for Examples 5-8

Reagents.

Curcumin, JSI-124 (cucurbitacin I) and LPS were purchased fromSigma-Aldrich (St Louis, Mo.) and dissolved in DMSO as stock solutions.A rabbit anti-Iba1 antibody that specifically recognizes microglia cellsand macrophages was purchased from Wako Chemicals (Richmond, Va.).Antibodies directly against total and phospho-Stat3 were purchased fromCell Signaling Technology Inc. (Danvers, Mass.). The followingfluorescent dye-conjugated Abs were obtained from e-Bioscience (SanDiego, Calif.): anti-CD11b, Anti-CD45.2, and anti-IL-1β.

Cell Lines.

The mouse (H-2^(b)) glioma cell line GL26 stably expressing theluciferase gene (GL26-Luc) and the BV2 microglia cell line were providedby Dr. Behnam Badie (Beckman Research Institute of City of Hope, LosAngeles), and maintained in RPMI 1640 media supplemented with 10%heat-inactivated FBS in a humidified CO₂ incubator at 37° C. Cell linesincluding 3T3L1, 4T1, CT26, A20, and EL4 were purchased from theAmerican Type Culture Collection (ATCC; Manassas, Va.) and culturedaccording to the protocols provided by the ATCC.

Preparation of Exosomes and Exosomal Curcumin (Exo-Cur) and JSI-124(Eva-JSI-124).

All exosomes used in this study were prepared according to the protocolsdescribed herein above. Microparticles were prepared from supernatantsof tumor cells grown to confluence (48 h). The supernatants weresequentially centrifuged at 500×g for 10 minutes and then at 1200×g for30 minutes. Microparticles were then pelleted at 10,000×g for 1 hour andwashed once in PBS. The concentration of exosomes and microparticles wasdetermined by analyzing protein concentration using the Bio-Rad proteinquantitation assay kit (Bio-Rad, Hercules, Calif.) with bovine serumalbumin serving as a standard. Both Exo-cur and Exo-JSI-124 wereprepared by mixing curcumin or JSI-124 with EL-4 exosomes in PBS. Afterincubation at 22° C. for 5 minutes, the mixture was subjected to sucrosegradient (8, 30, 45, and 60%, respectively) centrifugation for 1.5 hoursat 36,000 rpm. Exo-cur or Exo-JSI-124 was subsequently collected,washed, and resuspended in PBS. The concentration of curcumin or JSI-124in the complex was determined by HPLC analysis as also described hereinabove.

Animals.

C57BL/6j mice (H-2^(b)) were purchased from the Jackson Laboratory.Animals were housed in the animal facility at the University ofLouisville per an Institutional Care and Use Committee-approvedprotocol.

Intranasal Delivery of Exosomes, EXO-CUR and EXO-JSI-124 in Mice.

For intranasal administration of exosomes or exosome encapsulated drugs,C57BL/6j mice were anesthetized by inhalation with 2%-2.5% isofluraneand placed in a supine position in an anesthesia chamber. PBS (2 μl)containing exosomes (300 pmol/2 μl), or Exo-cur or Exo-JSI-124 wereadministered intranasally as drops with a small pipette every 2 min intoalternating sides of the nasal cavity for a total of 10 min. A totalvolume of 10 μl was delivered into the nasal cavity.

To determine the bioavailability of free curcumin and exosomal curcuminin vivo, two groups (five per group) of C57BL/6j mice were administrated1.5 nmol curcumin or Exo-cur intranasally. At 0, 3, 6, 12, and 24 hours,the olfactory bulb was removed and curcumin was extracted from thetissue as described herein above. The concentration of curcumin in theextracts was again determined by HPLC methodology. The extracts fromolfactory bulb of naive mice without treatment either mixed with a knownamount of curcumin or PBS were used as positive and negative controls,respectively.

To monitor the trafficking of exosomes administered intranasally,exosomes were first labeled using an Odyssey fluorescent dye IRDye800kit (LI-COR Biosciences, Lincoln, Nebr.) and a previously describedmethod (82). To localize EL-4 exosomes in brain tissue, the IRDye800CW-labeled EL-4 exosomes (10 μg/10 μl in PBS) were 1 administratedintranasally to C57BL/6j mice as described above. The mice were imagedover a 48-hour period using a prototype LI-COR imager (LI-CORBiosciences). For controls, mice (five per group) received nonlabeledEL-4 exosomes in PBS or free IRDye800 dye at the same concentration forIRDye800 dye labeled exosomes.

Identifying the Brain Cells Targeted by Exosomes AdministratedIntranasally.

Mice were administered intranasally PHK26 fluorescent dye labeledexosomes (10 μg/mouse in 10 μl PBS) using the method described above.After intranasal administration, mice were transcardially perfused withPBS followed by a 4% paraformaldehyde (PFA) solution at pH 7.4. Braintissue was postfixed overnight in 4% PFA and then cryopreserved inphosphate-buffered 30% sucrose. Brains were embedded in Tissue-Tek (OCTcompound, Sakura, USA) and kept at −20° C. overnight. Brain tissuesections were cut with a Cryostat (30 μm thick) and the tissue sectionsstored at −20° C. Immunofluorescent staining of microglia cells withrabbit anti-Iba1 antibody was carried out according to previouslydescribed procedures (83). Tissues evaluated for the presence of Iba1positive staining were assessed using a Zeiss LSM 510 confocalmicroscope equipped with a digital image analysis system (Pixera, SanDiego, Calif.).

Brain Tumor-Bearing Mice Model.

5×10⁴ GL26-Luc cells per mouse were intracranially injected using amethod described previously (84). In brief, using a Hamilton syringe(Hamilton Company, Reno, Nev.), 5×10⁴ GL26-Luc cells in 2 μl PBS werestereotactically injected through an entry site at the bregma ofanesthetized mice. Typically this procedure results in a 100% tumor takeand a median survival time of approximately 22 days after tumorimplantation. Tumor-bearing mice were treated intranasally for 12consecutive days with daily doses of 12.5 pmol Exo-JSI-124, orJSI-124-(12.5 pmol) or Exo-control in PBS or PBS-control. Treatment wasinitiated on day 3 after tumor cells were injected intracranially. Theinvestigators treating the animals were fully blinded with regard totreatment. All mice were monitored every day and euthanized when theyexhibited neurological symptoms indicative of impending death.

Monitoring the growth of injected tumor cells was accomplished byquantifying luciferase activity over a 15-day period post-tumor cellinjection using a previously described method (85) with minormodifications. In brief, prior to the imaging session, the mice receivedan intraperitoneal (IP) injection of D-luciferine, a luciferasesubstrate (150 mg/kg, Xenogen, Alameda, Calif.) dissolved in PBS. Themice were then anesthetized with 2% isoflurane in 100% oxygen at a flowrate of 2 mL/min. Images were collected using a high-sensitivity CCDcamera with wavelengths ranging from 300 to 600 nm with an exposure timefor imaging of 2 min. Regions of interest were analyzed for luciferasesignals using Living Image 2.50 software (Xenogen) and was reported inunits of relative photon counts per second. The total photon count perminute (photons per minute) was calculated (5 animals) using LivingImage software. The effects of treatment versus non-treatment on braintumor bearing mice was determined by dividing the number of photonscollected for treated mice by the number of photons collected foruntreated mice at different imaging time points. Results wererepresented as pseudocolor images indicating light intensity (red andyellow being the most intense) that were superimposed over grayscalereference photographs.

LPS Induced Brain Inflammation.

Bacterial lipopolysaccharide (LPS) mg/kg, Sigma-Aldrich) was injected IPinto C57BL/6j mice. Immediately after LPS injection, mice wereadministrated intranasally curcumin, exo-cur (1.5 nmol in 10 μl PBS), orEL-4 exosomes equal to the amount in exosomal curcumin. EL-4 exosomesand PBS served as controls. Two h after the treatments five mice fromeach group of mice (10 mice per group) were sacrificed and the skulls ofthe mice were removed, the exposed brains were photographed, and thebrains were subsequently fixed for analysis of apoptosis induction usinga method described previously (86, 87). Four hours after the treatments,the remaining mice (5 mice per group) in each group were sacrificed andbrain leukocytes were isolated. The percentage of activated microgliacells and apoptotic cells was determined by fluorescence-activated cellsorting (FACS) analysis of CD45.2⁺IL-1β⁺ cells and PI⁺ annexin Vpositive staining cells, respectively. Apoptosis in the brain was alsoevaluated by fluorescence using an in situ cell death detection kit(Roche) according to manufacturer's protocol. The expression of IL-1β inCD45.2⁺IL-1β⁺ cells was quantified by real-time PCR (88).

Experimental Autoimmune Encephalitis (EAE) Induction and Treatment withExo-Cur In Vivo.

EAE was induced in six-week-old female C57BL/6 mice using a proceduredescribed previously (67). Briefly, mice were primed subcutaneously inthe flanks with 150 μg of Myelin Oligodendrocyte Glycoprotein (MOG)35-55 peptide (Biosynthesis, Lewisville, Tex.) per animal. The peptidewas emulsified in complete Freund's adjuvant (CFA) containing 1 mg/ml ofMycobacterium tuberculosis H37RA (Difco, Detroit, Mich.). Two days laterthe mice were injected IP with 500 ng of Pertussis toxin (Alexis Corp.,San Diego, Calif.) in 100 μl of PBS. Mice (n=10) were treatedintranasally with daily doses of 1.5 nmoles of Exo-cur, or with Cur- orExo-controls in PBS or PBS-control for 26 consecutive days. Treatmentwas initiated on day 4 after mice were primed with MOG35-55 peptide. Themice were scored as follows: 0, no detectable signs of EAE; 1, completelimp tail; 2, limp tail and hindlimb weakness; 3, severe hindlimbweakness; 4, complete bilateral hindlimb paralysis; 5, total paralysisof both forelimbs and hindlimbs or death.

Isolation of Brain Leukocytes.

Brain leukocytes were isolated using a method described previously (89).In brief, mice were sacrificed by CO₂ asphyxia, then perfused throughthe left cardiac ventricle with PBS. Brains were minced mechanically andcells from each brain were resuspended in 70% Percoll (Sigma-Aldrich,St. Louis, Mo.), overlayed with 37 and 30% Percoll, and centrifuged for20 min at 500×g at 22° C. Enriched brain leukocyte populations wererecovered at the 70-37% Percoll interface. Quantification of subsetpopulations present in the isolated cells was determined by antibodystaining followed by FACS analysis (89) or western blot analysis of cellspecific proteins.

For FACS analysis of cell apoptosis, an annexin-V fluoresceinisothiocyanate/PI double-stain assay was performed according to themanufacturer's protocol (BioVision, Mountain View, Calif.). Briefly,leukocytes isolated from brain tissue were washed and resuspended in 500μl of binding buffer containing 5 μl of annexin-V fluoresceinisothiocyanate and 5 μl of PI. The cells were incubated for 5 minutes inthe dark at 22° C. Analysis was done immediately using a flow cytometer.

Western Blot.

Western blots were done as previously described (90). In brief, cellswere lysed and proteins of lysed cells were separated on 10%polyacrylamide gels using SDS-PAGE. Separated proteins were transferredto nitrocellulose membranes. The western blot was carried out with theanti-Stat3 and anti-phospho-Stat3 antibodies (Cell Signaling, Danvers,Mass.) or anti-β-actin antibody (Santa Cruz Biotechnology, Santa Cruz,Calif.).

Cytokine Assay.

Culture supernatants were assessed for mIL-IL-1β using an ELISA kit(eBioscience, San Diego, Calif.).

Quantitative Real-Time PCR (QPCR).

Relative quantification of select mRNA was performed using a CFX96Realtime System and SsoFast™ Evagreen® supermixture (Bio-RadLaboratories, Hercules, Calif.) according to the manufacturer'sinstructions. All primers were purchased from Eurofins MWG Operon(Huntsville, Ala.). Fold changes in mRNA expression between treatmentsand controls were determined the ΔCT method (91). Fluorescence thresholdcycle (C_(t)) values were calculated using SDS 700 System Software(Bio-Rad Laboratories, Hercules, Calif.). Results were normalized to theaverage C_(t) for the GAPDH and β-actin housekeeping genes run in theQPCR. ΔΔC_(t) values were calculated to determine expression changes.Differences between groups were determined using a two sided Student'st-test and one-way ANOVA. Error bars on plots represent +/− standarderror (SE), unless otherwise noted.

Statistical Analysis.

Survival data were analyzed by log rank test. Student's t test was usedfor comparison of two samples with unequal variances. One-way ANOVA withHolm's post hoc test was used for comparing means of three or morevariables.

Example 5—Intranasally Administered Exosomes Rapidly DistributesThroughout the Brains of Mice

To determine whether exosomes can be transported intranasally into thebrain, Odyssey 800 dye-labeled exosomes were administered tonon-tumor-bearing mice. Odyssey 800 dye-labeled exosomes (10 μg/10 μl)isolated from different types of cells (FIG. 7A) were administered asfive doses of 2-μl drops spaced 2 min apart and one 2-μl drop intoalternating sides of the nasal cavity with a small pipette. Mice wereeuthanized 30 min after intranasal delivery and their brains wereexamined for the presence of the exosomes using an Odyssey scanner.Fluorescent labeled exosomes were observed as being diffusely located inthe brain with their primary location being in the olfactory bulb,suggesting that translocation of exosomes to the brain occurred rapidly(FIG. 7A). In contrast, no microparticles larger than exosomes weredetected in the brain (FIG. 7A). Very little or no fluorescence wasdetected in the brain of mice intranasally administered PBS or free dye(FIG. 7A). These results indicate that particle size is a factor fortranslocation from the nasal region to the brain. No apparent toxicityor behavioral abnormalities were observed in any of the mice during andafter (30 days) the experiment. The distribution of intranasalIRDye800-labeled exosomes in the brain was next investigated at varioustimes after intranasal delivery of EL4 exosomes. Mice were given 10 μgIRDye800-labeled EL4 T cell derived exosomes over a 10-min time periodas noted above, and then euthanized 3 to 48 h later. Fluorescence wasstronger throughout the brain at 3 h after intranasal delivery, andremained visible at the olfactory bulb region of the brain 24 h afterdelivery (FIG. 7B). The animals did not exhibit any apparent toxicitiesor behavioral abnormalities during and after the course of thisexperiment.

Next, the capacity of exosomes to deliver curcumin to the brain wasdetermined. The results of the quantification of curcumin intranasallydelivered by EL4 exosomes (Exo-Cur) revealed that the curcumin reachedpeak concentrations 1 h after intranasal administration, and was stilldetectable in the olfactory bulb region within the first 12 h after asingle intranasal administration of Exo-cur (FIG. 8A). Repeatedadministration of Exo-cur every 12 h maintained the curcuminconcentration at an average of 5.6±1.2 nmol/g of brain tissue in theolfactory bulb region (FIG. 8B). Collectively, these data indicate thatexosomes can be used as a novel non-invasive vehicle for delivery oftherapeutic agents to the brain.

Example 6—Intranasally Delivered Exosomes Preferentially Accumulate inMicroglia Cells

To further identify specific targeting of cells by exosomes, in vivobiodistribution of fluorescent dye PKH26-labeled EL-4 exosomes wasconducted. Double fluorescence positive cells evidenced by doublepositive PKH26⁺Iba-1⁺ cells (Iba-1 is a specific marker for microgliacells) were visible in brain microglia cells 15 min after intranasaldelivery of exosomes, (FIG. 9A). Within an hour after injection, morethan 80% of Iba-1⁺ microglia cells were PHK26 positive (FIGS. 9A and9B), indicating that the injected exosomes were taken up by themicroglia cells.

Example 7—Exosome Encapsulated Curcumin Inhibits LPS Induced BrainInflammation and MOG Induced Autoimmune Responses in an EAE Model

Microglia cells have been shown to play a crucial role in braininflammation. To determine whether the exosomes are functioning as adelivery vehicle to carry anti-inflammatory drugs, such as curcumin, andtherefore treat brain inflammatory diseases, two independent diseasemodels were tested. In the first model, intranasal administration ofexosome encapsulated curcumin was used for treating LPS-challenged mice.The results indicate that 4 h after LPS challenge, mice receivingintranasal exosomal curcumin have reduced brain inflammation with lessvisible blood vessels (FIG. 10A) in comparison to other mice treatedwith either curcumin alone at the same dose as Exo-cur or exosomesalone. FACS analysis further indicated that the number of activatedinflammatory microglia cells (CD45.2⁺IL-1β⁺) was significantly reducedin the brain of mice that were treated intranasally with Exo-cur incomparison to the other treatments listed in FIG. 10B. The reduction ofIL-1β in CD45.2 microglia cells was further confirmed by real-time PCR(FIG. 10C). The results of TUNEL staining of brain tissue indicated thatExo-cur treatment led to an increase in the number of apoptotic cells inmice administrated Exo-cur intranasally (FIG. 10D). Co-staining with amicroglia cell-specific antibody (Iba-1 clone) (FIG. 10E) indicated thatdouble positive cells (Tunel⁺IBA-1⁺) were microglia cells. The inductionof apoptosis was further confirmed by the results of FACS analysis (FIG.10F). The induction of apoptotic microglia cells correlated with theconcentration of curcumin detected in the brain of mice treated withExo-cur (FIG. 10G).

To further determine if intranasal delivery of Exo-cur could preventinflammation related brain autoimmune disease, MOG induced experimentalautoimmune encephalomyelitis (EAE) in mice was utilized. EAE was inducedin six-week-old female C57 BL/6 mice by immunization with MOG 35-55 asdescribed previously (67). Exo-cur was administrated intranasally dailyusing the protocol described above and was initiated on day 4 afterimmunization with the MOG peptide until mice were sacrificed at day 35post immunization. Disease severity was scored based on the method asdescribed previously (67). The score of PBS, exosomes only, or curcuminonly groups of EAE mice was 3.83±0.22, 3.70±0.31, and 3.60±0.12,respectively. The disease severity in Exo-cur treated mice wassignificantly reduced with a maximal disease severity score of 1.53±0.41(FIG. 11A). Real-time PCR analysis also demonstrated that the expressionof IL-1β in CD45.2 microglia cells was decreased significantly in theExo-cur treated mice (FIG. 11B) in comparison with control groups.

Example 8—Intranasal Delivery of an Exosome Encapsulated STAT3 InhibitorReduces GL26 Tumor Growth

Signal transducer and activator of transcription 3 (Stat3) is activatedconstitutively in many types of human cancers and plays a critical rolein tumor growth including microgliomas. Microglia cells, the residentmacrophages of the brain, have been known to play a critical role in theprogression of brain microglioma. To determine if exosome-encapsulatedStat3 inhibitors could be used to reduce the progression of brainmicrogliomas, groups of mice bearing intra-cerebral tumors were treatedwith exosome-encapsulated Stat3 inhibitor JSI-124 (12.5 pmol/10 μl) orexosomes only, JSI-124 only, or PBS as controls. Mice were treated everyother day for 15 days beginning on day 3 after tumor cells wereimplanted. The amount of JSI-124 administered was based on the lack ofany evidence of toxicity or behavioral abnormalities in the mice.Imaging data showed a statistically significant decrease inbrain-associated photons in Exo-JSI-124 treated mice when compared tocontrols (FIG. 12A), and determined on day 9 after tumor cells injected.Survival times of PBS-, exosomes- or JSI-124-control animals ranged from20 to 25 days. In contrast, Exo-JSI-124 treatment significantlyprolonged the survival of mice on the average of 44.5 days (p<0.011)(FIG. 12B). Moreover, two of the ten Exo-JSI-124 treated mice were aliveand showed no neurological symptoms at day 90 when they were euthanized.There was no evidence of tumor at the original implantation site inthese two mice. None of the Exo-JSI-124-treated animals exhibitedevidence of toxicity or behavioral abnormalities during and after the15-day treatment period. To further investigate if the observed effectwas due to inhibition of Stat3 activity in the Exo-JSI-124 targetedcells in the brain, the activity of Stat3 in brain CD45.2⁺ cells wasquantitatively determined by western blot analysis of pStat3. Theresults indicated that Exo-JSI-124 treatment led to the selectivereduction of pStat3 in CD45.2⁺ microglia cells (FIG. 12C). The reductionof Stat3 was also correlated with a decrease in the expression of bothIL-1β and IL-6 in CD45.2⁺ microglia cells (FIG. 12D). Collectively,these data indicated that Exo-JSI-124 is selectively taken up bymicroglia cells and subsequently inhibits the expression of inflammatorycytokines such as IL-1β and IL-6.

Discussion of Examples 5-8

The blood-brain barrier has been an obstacle to the development of CNStherapeutics, impeding clinical use of otherwise promising therapeuticagents in the treatment of many brain neuron disorders, whereinflammation plays a causative role. Despite these previous obstacles,the experiments described in the foregoing examples 5-8 examined a novelapproach for intranasal delivery of therapeutic agents to the brain. Theresults of those experiments indicated that anti-inflammatory agentslike curcumin or JSI-124 were effectively delivered to the brain byexosomes without observable side effects. Furthermore, microglia cellswere identified as being preferentially targeted by exosomes. Thesuccessful delivery and therapeutic effects of curcumin or JSI-124loaded exosomes was demonstrated in three independent mouse models,i.e., a LPS induced brain inflammation model, MOG induced EAE autoimmunedisease, and a GL26 implanted brain tumor model. Microglia cells arewell-known to play an essential role in many inflammatory related braindiseases. The accumulation of myeloid or microglia cells in the brainhas been implicated in the promotion of brain tumor growth andprogression of brain autoimmune diseases, such as EAE in mouse modelsand in humans (68-79). In the experiments described in Examples 5-8, itwas found that intranasal administration of Exo-cur or Exo-JSI-124 ledto a significant reduction in the number of microglia cells and aconcomitant reduction in disease progression in all three models wetested. These findings are also consistent with the results described inExamples 1-4 showing that inflammatory cells such as CD11b⁺Gr-1⁺ cells,can be deleted specifically by curcumin encapsulated in exosomes. InExamples 1-4, it was demonstrated that mice treated with Exo-cur areprotected completely against LPS-induced septic shock and that theprotective mechanism is associated with the ability of the Exo-cur tospecifically target myeloid cells. Without wishing to be bound by anyparticular theory, it was thus thought that the strategy of deliveringexosome encapsulated drugs to the brain via intranasal administrationcould potentially improve the direct delivery of drugs to the CNS withthe advantages of target specificity and administration in anon-invasive manner. In this regard, the foregoing data show thatcurcumin encapsulated in exosomes not only targets to inflammatorycells, i.e., microglia cells, but reaches the CNS in sufficient quantityby this route to be effective.

In summary, the foregoing results indicate that directintranasal-to-brain transport is feasible. Additionally, rapid movementof exosomes into the brain was found within 1 hour. This finding isconsistent with the extraneuronal pathway that has been proposed fortransport of therapeutic agents from the nasal cavity to the brain(52-53). Transport occurs along the olfactory pathway and likelyinvolves extracellular bulk flow along perineuronal and/or perivascularchannels, which delivers drug directly to the brain parenchyma. Deliveryalong the extraneuronal pathway is likely not receptor-mediated andrequires only minutes for a therapeutic agent to reach the brain;whereas, delivery via an intraneuronal pathway along the primaryolfactory sensory neurons involves axonal transport and requires severaldays for the drug to reach different areas of the brain (80, 81).

Example 9—Preparation of Exosomes Incorporating Additional PhytochemicalAgents or Chemotherapeutic Drugs

Exosomes including the phytochemical agents resveratrol, baicalein,equal fisetin, quercetin, or exosomes including the chemotherapeuticagents retinoic acid, 5-fluorouracil, vincristine, actinomycin D,adriamycin, cisplatin, docetaxel, doxorubicin, or taxol are prepared bymixing the one or more of the phytochemical agents or chemotherapeuticagents with exosomes in PBS. After incubation at 22° C. for 5 minutes,the mixture is subjected to a sucrose gradient (8, 30, 45, and 60%,respectively) and then centrifugation for 1.5 hours at 36,000 rpm. Theexosomal phytochemical agents or chemotherapeutic drugs band in thesucrose gradient between 45 and 60%, and are subsequently collected,washed, and dissolved with PBS. The aliquots are then stored at −80° C.until use.

Example 10—Use of Exosomes or Exosomal Phytochemical Agents orChemotherapeutic Agents for Treatment of Colon Cancer

Mice, 6-8 wk of age, are injected intraperitoneally with 10 mg/kgazoxymethane (AOM) in 0.2 ml PBS. 1 week after AOM administration,dextran sulfate sodium (DSS) at 2% is administered in the drinking waterfor five consecutive days. Thereafter, mice received reverse osmosiswater. Up to four DSS cycles are administered with intervals of 16 d onwater between cycles. The mice are gavage-administrated with exosomes orexosomal phytochemical agents or chemotherapeutic agent in 0.3 ml PBS.Mice are treated daily for 3 weeks. Mice are monitored for body weight,rectal prolapse, diarrhea, and macroscopic bleeding, as well as occultblood by hemoccult (Beckman Coulter, Brea, Calif.). After the mice aresacrificed, the colons are resected, flushed with PBS, openedlongitudinally, and measured. Polyps are then counted using astereomicroscope and colon sections are fixed in formalin or snapfrozen. Upon analysis of the results of these experiments, it isobserved that mice administered exosomal phytochemical agents orchemotherapeutic agents have a lower number of polyps as compared tocontrol mice, indicating that the exosomal phytochemical orchemotherapeutic agents can successfully be administered as part of amethod for treating colon cancer.

Throughout this document, various references are mentioned. All suchreferences are incorporated herein by reference, including thereferences set forth in the following list:

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It will be understood that various details of the presently disclosedsubject matter can be changed without departing from the scope of thesubject matter disclosed herein.

What is claimed is:
 1. An exosomal composition comprising a therapeuticagent, the therapeutic agent being encapsulated by an exosome derivedfrom a plant, wherein the exosome is isolated from the juice of a fruitof the plant, and further wherein the fruit is selected from the groupconsisting of a grape, a grapefruit, and a tomato.
 2. The exosomalcomposition of claim 1, wherein the therapeutic agent is an siRNA. 3.The exosomal composition of claim 1, wherein the exosome specificallybinds to a target cell or tissue.
 4. The exosomal composition of claim1, further comprising a pharmaceutically-acceptable vehicle, carrier, orexcipient.
 5. The exosomal composition of claim 1, wherein thecomposition is formulated for intranasal, oral, or intratumoraladministration.
 6. An exosomal composition comprising a therapeuticagent, the exosomal composition being produced by a process comprising:(a) isolating one or more exosomes from the juice of a fruit of a plantselected from the group consisting of a grape, a grapefruit, and atomato; and (b) incubating the one or more exosomes with a therapeuticagent, wherein the one or more exosomes encapsulate the therapeuticagent.